The prevalence of non-alcoholic fatty liver disease (NAFLD) and its severe phenotype, non-alcoholic steatohepatitis (NASH), are increasing significantly in the United States in the adult and pediatric population. NAFLD is associated with obesity and metabolic syndrome and its prevalence has increased in parallel to the prevalence of obesity and type-2 diabetes. The development of NAFLD, its different phenotypes, and the heterogeneity of disease progression are not completely understood. Recent evidence suggests that there is an association between intestinal microbial colonization (the intestinal microbiome) and obesity in humans and in animal models. In addition, there is evidence of abnormalities of bacterial colonization, and intestinal bacterial product induced inflammation associated with NAFLD and progression to NASH. The goal of this proposal is to investigate the composition of the intestinal microbiome in pediatric patients with obesity and obesity plus NAFLD, and determine the relationship between alterations in the intestinal microbiome, immune activation, and the development of NAFLD. We hypothesize that alterations in the intestinal microbiome are associated with increased immune activation and the development of NAFLD. The focus of aim 1 is to ascertain well-phenotyped pediatric subjects with NAFLD and a well-characterized pediatric obese control group, and evaluate their clinical status, and extent of systemic inflammation. Pediatric participants with NAFLD will be recruited in collaboration with Dr. Jeffrey Schwimmer and the existing NIDDK-sponsored multicenter NASH Clinical Research Network (NASH CRN) and through the adolescent obesity program at UCSD. Obese controls will be evaluated for liver steatosis by magnetic resonance spectroscopy (MRS). Aim 2 focuses on the study of the intestinal microbiome of children with NAFLD and in obese controls. High through put 16S rDNA analysis, shotgun sequencing, metagenome/metatranscriptome analysis of bacterial genomic DNA/RNA isolated from fecal specimens will be used to identify the characteristic microbiome of each individual within the 2 different study groups. Aim 3 will focus on the analysis of the relationship between NAFLD, systemic inflammation, and the intestinal microbiome. Characteristics of the intestinal microbiome indicative of the development of NAFLD will lead to an increased understanding of the disease process. Greater understanding of the role of the intestinal microbiome in the development and progression of NAFLD could lead to the identification of novel biomarkers to predict susceptibility to NAFLD in populations with obesity, and also to novel interventions in the prevention and treatment of this disease through the manipulation and modulation of the intestinal microbiome or its functional metabolic capacity through the use of prebiotics, probiotics, antibiotics, or via other newly revealed mechanisms. PUBLIC HEALTH RELEVANCE: The incidence of non-alcoholic fatty liver disease (NAFLD) is increasing significantly in the United States in the pediatric population in association with increasing prevalence of obesity and type-2 diabetes, and is rapidly becoming a major reason for liver failure and transplantation. Recent evidence suggests that there is an association between intestinal microbiome (the bacteria and microbes that normally colonize the human GI tract) and the development of NAFLD. The goal of this study is to investigate the composition of the intestinal microbiome in obese pediatric patients with and without NAFLD, to determine whether there are differences in their intestinal bacteria that could relate to the development of fatty liver and liver inflammation. These studies will lead to a greater understanding of the development and progression of NAFLD and may lead to novel approaches for the treatment of a disease that is becoming an increasing public health concern.